Diffuse axonal injury (DAI) is a common feature of many forms of traumatic brain injury (TBI) and its occurrence has been linked with the morbidity and mortality associated with TBI. Historically, it was assumed that the shear and tensile forces of injury immediately tore axons at the moment of impact. More contemporary studies, however, have demonstrated that this is typically not the case; rather, it has been shown that the traumatic episode causes focal change in the axon that impairs axoplasmic transport, leading to swelling and disconnection. The genesis of these intra-axonal abnormalities appears to lie in diverse forms of intra-axonal cytoskeletal change involving either cytoskeletal misalignment or compaction. The goal of this application is to characterize these initiating cytoskeletal events while providing insights into the mechanisms involved in their genesis. Scientifically constrained by the problems inherent in study diffusely injured axons scattered in fields of other structurally intact survivors, we have developed, in our estimation, novel approaches to address these issues. Using a well-characterized rodent model of inertial impact, we will follow these intra-axonal events over time using extracellular markers to delineate concomitant changes in axolemmal permeability. Parallel populations of animals will be probed at the light and electron microscopic level with antibodies targeting epitope specific cytoskeletal constituents primarily focusing on neurofilaments and their sidearms in a phosphorylation-dependent and independent fashion. These studies will be also interfaced with detailed, computer-assisted, quantitative ultrastructural analyses to determine if a change in phosphorylation state is pivotal for initiating intra-axonal cytoskeletal change. In concert with the above studies, other antibodies will be employed to study the potential for calcium-mediated proteolysis. The potential involvement of calcium will be also explored through the use of calcium chelators. Further, all of the cytoskeletal changes will be compared and contrasted to those seen through the use of antibodies targeting amyloid precursor proteins which are known light microscopic markers of human axonal injury. Upon completion of these studies in rodents, comparable studies will be repeated in a pig model of TBI whose gyrencephalic features allow for replication of most of the important features of human DAI. Lastly, many of these experimental approaches will be pursued in tissue obtained from human postmortem analysis in order to better ascertain if the events described in experimental animals are operant in humans. The work proposed in this application should significantly expand our understanding of traumatically induced axonal injury, Such information may prove helpful for designing more rationale therapeutic interventions.